Annals of the Rheumatic Diseases 1997;56:135–139 135

Use of monoclonal antibodies to detect disease Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from associated HLA-DRB1 alleles and the shared epitope in rheumatoid arthritis

Ian Wicks, GeoV McColl, Angela D’Amico, Loretta Dougherty, Brian Tait

Abstract earlier and more aggressive therapeutic Objective—To use a panel of monoclonal intervention. Early stratification of patients antibodies (Mab) which recognise HLA who are at risk for severe disease is an class II alleles associated with rheumatoid important clinical issue for these reasons. arthritis for fluorescence activated cell Many studies have confirmed an association sorter (FACS) analysis of peripheral blood between rheumatoid arthritis and a restricted mononuclear cells (PBMNC) from pa- number of alleles at the highly polymorphic tients with early and established rheuma- HLA-DRB1 locus (primarily DRB1*0401, toid arthritis and to compare these results *0404, and *0101).23 Each of these alleles against DNA oligotyping of HLA class II shares sequence homology in the third molecules in the same patients. hypervariable region of the â chain, which is Methods—27 patients (18 from an early referred to as the shared epitope. In a study of arthritis clinic, nine with established 201 patients with rheumatoid arthritis, who rheumatoid arthritis) were studied using were unselected for disease severity, the both techniques. PBMNC were stained presence of a single copy of *0401, *0404, or with Mab which recognise the shared *0101 was associated with an odds ratio for epitope, the HLA-DRB1*04 molecule and rheumatoid arthritis of 3.5.4 Two identical its *0401, *0404 subtypes in the presence of copies of these alleles gave an odds ratio of 7.8 bound peptide. Mab stained cells were and this increased to 25.6 with *0401/*0404 analysed by FACS. Genomic DNA was compound heterozygosity. Similar results were prepared from PBMNC and used for DNA obtained from an analysis of 120 patients pre- oligotyping and sequencing by standard senting with early arthritis.5 In this study, 10/11 methods. compound heterozygotes (*0401/*0404) dev- Results—FACS analysis of Mab stained eloped erosions at one year. Conversely, PBMNC gave identical results to those patients without the HLA-DRB1 susceptibility obtained by DNA oligotyping in 26/27 alleles are at low (10-20%) risk for progressive patients. The antibodies identified the rheumatoid disease.23 These data raise the http://ard.bmj.com/ shared epitope in 14/14 cases and the pres- possibility of using HLA typing, together with ence of an HLA-DRB1*04 molecule in other clinical and laboratory indices, to 12/12 cases. HLA-DRB1*0404 was identi- compile a profile of the patient with early fied in 4/4 cases. HLA-DRB1*0401 was inflammatory arthritis who is at risk for more identified in 5/6 cases. One patient severe rheumatoid disease. oligotyped as HLA-DRB1*0401, but HLA analysis has traditionally been

Reid Rheumatology consistently failed to react with the *0401 performed in the tissue typing laboratory, on September 29, 2021 by guest. Protected copyright. Laboratory, Burnet Mab. DNA sequencing of the second exon initially using serological and cellular typing Clinical Research of the HLA-DRB1*0401 allele in this reagents and more recently with molecular Unit, Walter and Eliza Hall Institute of patient confirmed a normal HLA- techniques. The standard method for Medical Research, DRB1*0401 genotype. determining HLA-DR and its subtypes now Parkville, Victoria, Conclusions—FACS analysis of PBMNC involves isolation of genomic DNA from Australia I Wicks stained with Mab recognising the shared peripheral blood cells, the polymerase chain G McColl epitope and rheumatoid arthritis associated reaction (PCR) with sequence specific A D’Amico HLA susceptibility molecules provides a oligonucleotide primers and dot blotting, L Dougherty rapid, reliable, and more accessible followed by hybridisation with probes directed Tissue Typing alternative to DNA oligotyping. The appar- at hypervariable region sequence diVerences. Laboratory, Royal ent discordance between phenotypic and These molecular techniques, which are Melbourne Hospital, genetic analysis of HLA-DRB1*0401 in one generally carried out in the context of HLA Parkville, Victoria, Australia patient, may reflect variability in HLA- matching for organ transplantation, require B Tait DRB1*0401 gene expression or in class II specialised expertise and equipment and strin- peptide presentation. gent quality control. While HLA typing has Correspondence to: Dr Ian Wicks, Reid been used as a research tool in the study of Rheumatology Laboratory, (Ann Rheum Dis 1997;56:135–139) rheumatoid arthritis, it has not been generally Burnet Clinical Research Unit, Walter and Eliza Hall accessible as an aid to clinical management. Institute of Medical Rheumatoid arthritis is a common but Recently, monoclonal antibodies (Mab) Research, PO Royal Melbourne Hospital, Victoria clinically heterogeneous disease. Recent work which recognise HLA-DR4, its subtypes *0401 3050, Australia. suggests that much of the joint damage in more and *0404, and the shared epitope severe cases occurs in the first few years of the (QKAA/QRAA) have been generated.67 These Accepted for publication 1 4 September 1996 disease. Accordingly, there is a trend towards antibodies are conformationally dependent 136 Wicks, McColl, D’Amico, Dougherty, Tait

and require peptide loading of the MHC mol- by double staining (figure). Increased B cell Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from ecule.6 Fluorescence activated cell sorter fluorescence in the phycoerythrin channel was (FACS) analysis of peripheral blood cells usually obvious from inspection, but quadrants potentially oVers a faster and more accessible were set on all samples, using goat anti-mouse method for identifying patients carrying (GAM) conjugated phycoerythrin alone rheumatoid arthritis associated HLA-DRB1 (negative control) and NFLD.M6 (positive alleles early in the course of the disease. In this control) to estimate staining in the phycoeryth- study, we compared the results of FACS analy- rin channel. The following formula was used to sis and genotyping in 27 patients, 18 of whom quantify fluorescence in the phycoerythrin came from an early inflammatory arthritis channel: clinic and nine had established rheumatoid arthritis. (%Test) - (%Negative control) × 100 (%Pos control) - (%Neg control) Methods SUBJECTS A figure of greater than 20% was recorded as Eighteen consecutive patients, all of whom ful- positive staining. FACS results were analysed filled the 1987 revised American Rheumatism without knowledge of the genotype. Association (ARA) criteria for rheumatoid arthritis, were enrolled from a study of early HLA DNA OLIGOTYPING inflammatory polyarthritis conducted at the DNA was isolated using the method of Miller Royal Melbourne Hospital (RMH). Patients et al 8 from the buVy coat of anticoagulated came from the community of suburban blood samples. Genomic DNA was amplified Melbourne. Nine patients with established by the PCR, dot blotted, and probed with dig- rheumatoid arthritis—drawn from the RMH oxygenin labelled (Boehringer Mannheim, outpatient rheumatology clinic—were also Germany) oligonucleotides specific for HLA- studied. DRB1 variable sequences. A two step procedure was used, consisting of PCR ampli- ANTIBODY STAINING fication of the DRB1 second exon using Peripheral blood was collected into generic primers and hybridisation with probes heparinised tubes and the mononuclear cells providing a serologically equivalent DR assign- isolated on a LeucoSep Ficoll density gradient ment (including DR4 and DR0101/0102). (Oropharma AG, Zurich). Peripheral blood Secondly, primers amplifying only the DR4 mononuclear cells (PBMNC, 2 × 106) were group of alleles were used in the PCR with stained with 30 µl of irrelevant mouse genomic DNA and the product probed with a immunoglobulin, Mab NFLD.D1 (anti-DR4; series of oligonucleotides which defined B1*04), Mab NFLD.D2 (anti-shared epitope *0401-*0422. A similar procedure was used to QKRAA/QRRAA), Mab NFLD.D11 (anti- define the DR1 alleles. Following hybridisation DR4Dw4; B1*0401), Mab NFLD.D13 and stringent washing, bound probes were (anti-DR4Dw14; B1*0404), or NFLD.M6 detected using an anti-digoxygenin-alkaline http://ard.bmj.com/ (Mab to monomorphic determinant on phosphatase conjugate and chemiluminescent HLA-DR; positive control) (supplied as super- substrate (Boehringer Mannheim, Germany). natants by Terra Nova Biotech, Newfound- Hybridisation signals were visualised by land, Canada). In cellular enzyme linked exposure to x ray film (Kodak XAR-5). immunosorbent assays (ELISA), the NFLD.D2 also showed limited binding to QARAA 7 HLA-DR4 1 SEQUENCING

containing alleles. Fluorescein isothiocyanate Primers flanking the second exon of the HLA- on September 29, 2021 by guest. Protected copyright. (FITC) conjugated Mab anti-CD3 (1:40) DR4 1 gene were used to PCR amplify a 300 (Dako, Denmark) was added to all tubes in base pair fragment from the genomic DNA of order to demarcate clearly the HLA class II patient 15. The PCR product was purified with expressing B cells. Cells were held for 20 min- a High Pure PCR product purification kit utes on ice and washed once through fetal calf (Boehringer Mannheim, Germany) and serum. Phycoerythrin conjugated goat anti- sequenced in both directions using a Taq Dye mouse Mab was added (Terra Nova Biotech, deoxy terminator cycle sequencing kit (Perkin Newfoundland, Canada) and cells held on ice Elmer, CA, USA) and an ABI automated for 30 minutes. Cells were washed once and DNA sequencer (Perkin Elmer, CA, USA). resuspended in 2% paraformaldehyde in phos- phate buVered saline before flow cytometry. Results FLOW CYTOMETRY The peripheral blood mononuclear cells from A Becton Dickinson FACScan and FACScan 18 patients with early inflammatory arthritis Research software (Becton Dickinson, CA, and nine patients with established rheumatoid USA) were used for analysis of Mab stained arthritis were stained with antibodies recognis- cells. Lymphocytes were separated from ing HLA-DRB1*04, its variants *0401 and monocytes and contaminating granulocytes *0404, and the shared epitope itself. Genomic using side-scatter and forward-scatter param- DNA was extracted from the same sample and eters. Fluorescence in the FITC channel was typed using PCR and sequence specific plotted against phycoerythrin for the gated oligohybridisation with DNA probes. The lymphocyte population. T cells were clearly figure shows representative FACS profiles and separated from the HLA class II positive B cells corresponding HLA genotypes. Increased Monoclonal antibodies in rheumatoid arthritis 137 Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from *0401+ *0101+ *0404+

Negative control

B1*04

Shared epitope

B1*0401 http://ard.bmj.com/

B1*0404 on September 29, 2021 by guest. Protected copyright. PE

FITC Two-colour FACS profiles of gated PBMNC stained with FITC conjugated murine anti-CD3 (X axis) and murine anti-HLA-DRB1*04, shared epitope, *0401, *0404, or negative control antibodies (Y axis). Cells were then washed and stained with phycoerythrin conjugated goat anti-mouse antibody. Genotypes (*0401, *0101, *0404) are listed above the figure. Anti-CD3 coated T cells also stain with the goat anti-mouse secondary antibody and are then clearly demarcated as a “double positive” population in the upper right quadrant. Positive HLA class II staining on B cells (in the lower left quadrant) is noted as a shift in fluorescence in the phycoerythrin channel.

fluorescence in the phycoerythrin channel the patients with early inflammatory arthritis (indicating positive staining) was usually obvi- (patients 1-18). Patient 15 oligotyped as HLA- ous on inspection, but quadrants were carefully DRB1*0401/*0101, but failed to react with the set on all samples, using the secondary *0401 antibody. Both the oligotyping and the antibody GAM-PE alone (negative control) FACS analysis were repeated on this patient and the Mab NFLD.M6, which recognises a and gave the same results. Sequencing of the non-polymorphic determinant on HLA-DR as second exon of the HLA-DRB1*04 allele using a positive control, to estimate staining in the genomic DNA as the substrate confirmed a phycoerythrin channel. As shown in the table, normal *0401 genotype in this patient. The the panel of Mab correctly recognised the results of antibody staining were equivalent to relevant cell surface products encoded by the HLA-DR oligotyping in nine patients (patients corresponding HLA genotypes in all but one of 19-27) with established rheumatoid arthritis. 138 Wicks, McColl, D’Amico, Dougherty, Tait

By FACS analysis, there was no cross reactivity modifying antirheumatic drugs (DMARD) Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from of the NFLD.D2 Mab with alleles which might and potential DMARD induced downregula- bear the QARAA epitope. tion of HLA class II expression. NFLD.D11 reactivity has been shown to be critically influ- Discussion enced by the presence of bound peptide in the Several recent studies have clearly established peptide binding site of the class II HLA 6 that HLA-DRB1 status contributes to the molecule, although the peptides involved are severity and outcome of rheumatoid unknown. Antibody binding in this system arthritis4 9 10–11 and is a useful prognostic marker could be influenced by the nature of the in early disease.5 However, for a variety of rea- peptides available for presentation. Our sons, HLA typing in rheumatoid arthritis has HLA-DRB1*0401/*0101 patient whose generally only been available in the research PBMNC failed to stain with NFLD.D11 may setting. Most tissue typing laboratories are have a disorder of peptide processing or necessarily preoccupied with typing for organ loading in the HLA class II pathway. However, transplantation and it is doubtful that even the positive staining for *0101 would argue limited HLA analysis would be routinely avail- against a generalised peptide processing abnor- able for patients with early inflammatory mality in this patient and might suggest arthritis. Ease of access and turnaround time reduced expression of the *0401 molecule from the laboratory are also important issues in itself. Variable transcription of HLA genes due clinical practice. to promoter polymorphisms has been 12 13 The present study was undertaken to evalu- described previously and is being further ate the performance of a panel of Mab which examined in our patient. recognise the HLA-DRB1 subtypes associated FACS analysis of Mab stained PBMNC is with rheumatoid arthritis. There was concord- rapid and the results were usually available ance between the results of FACS analysis of within a day of blood collection. Use of the Mab stained PBMNC s and DNA oligotyping antibodies and interpretation of results in 26/27 cases. The antibodies identified the requires a FACS machine and a skilled opera- shared epitope in 14/14 cases and the presence tor, although both of these are now commonly of the HLA-DRB1*04 molecule in 12/12 available in routine pathology laboratories. A cases. All four HLA-DRB1*0404 positive twofold increase in antibody staining due to patients were correctly identified. Five out of DNA homozygosity (as occurred with patients six *0401 positive patients were correctly iden- 12 and 18 in our study) is not readily tified. One patient genotyped as HLA- observable from the FACS profile and this is a DRB1*0401, but failed to react with the *0401 relative disadvantage of the Mab method. A Mab (NFLD.D11). A similar discrepancy “double dose”of the rheumatoid arthritis asso- between HLA-DRB1*0401 genotype and ciated susceptibility alleles is associated with 10 NFLD.D11 Mab reactivity has been encount- more severe disease and is detected by DNA ered in one other patient, but would appear to oligotyping. One possible use of the Mab method would therefore be to screen shared be unusual (Marshall WH, personal communi- http://ard.bmj.com/ cation). There are several possible explanations epitope positive patients in order to select those for this observation. All patients with needing DNA oligotyping. However, com- established rheumatoid arthritis were correctly pound heterozygosity (HLA-DRB1*0401/ typed in spite of the long term use of disease *0404) seems to carry the worst prognosis in rheumatoid arthritis4511 and this phenotype is Results of FACS analysis with the HLA-DR Mabs compared with the genotype as readily identifiable using NFLD.D11 and determined by DNA oligotyping in 18 patients (1–18) with early inflammatory arthritis NFLD.D13. We correctly identified the one and in nine (19–27) with established rheumatoid arthritis HLA-DRB1*0401/*0404 compound hetero- on September 29, 2021 by guest. Protected copyright. Shared zygote (patient 19) in our study using the Mab Patient B1*04 epitope B1*0401 B1*0404 Genotype panel. 1 + + + − 0401,7 Epidemiological studies using DNA oligo- 2 −−− − 2,10typing have established that several HLA class 3 + + − − 3,0405 II DRB1 alleles, and in particular the shared 4 + + − − 0403,0102 5 −−− − 7,13epitope coded by these alleles, exert an impor- 6 −−− − 2,3tant influence on the severity of rheumatoid 7 + + − + 0404,2 8 −−− − 3,11arthritis. DNA oligotyping remains the gold 9 − + − − 0101,2 standard for determining HLA status, but is 10−−− − 2,3not widely accessible and therefore has had 11 − − − − 11,2 12 + + + − 0401,0401 limited impact on clinical management in dis- 13 − − − − 07,0103 eases like rheumatoid arthritis. We believe sim- 14−−− − 2,3plified methods for detecting the shared 15 + + − − 0401,0101 16−−− − 2,3epitope itself and rheumatoid arthritis 17−−− − 3,13associated HLA subtypes—such as the one 18 + + + − 0401,0401 outlined in this paper—will aid in the prompt 19 + + + + 0401,0404 20 + + + − 0401,0101 identification of patients with early inflamma- 21 − + − − 0101,0103 tory arthritis who are at greater risk for severe 22 + + − + 0404,8 rheumatoid disease. 23 + + − + 0404,3 24 + + − − 0408,0101 25−−− − 3,11We thank Terra Nova Biotechnology for kindly supplying the 26−−− − 2,7antibodies used in this study and the rheumatologists who 27−−− − 2,8allowed us to study their patients. We thank Michael Varney and Carmel Kanaan from the Tissue Typing Laboratory of the Monoclonal antibodies in rheumatoid arthritis 139

Royal Melbourne Hospital for excellent technical assistance and 7 Drover S, Karr RW, Fu XT, Marshall WH. Analysis of Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from Peter McNair for help with sample collection. We gratefully monoclonal antibodies specific for unique and shared acknowledge the support of the Reid Charitable Trusts and the determinants on HLA-DR4 molecules. Hum Immunol Australian National Health and Medical Research Council. 1994;40:51–60. 8 Miller SA, Dykes DD, Ploesky HF. A simple salting out pro- cedure for extracting DNA from human nucleated cells. 1 Fuchs HA, Pincus T. Radiographic damage in rheumatoid Nucleic Acids Res 1988;16:1215. arhtritis: description by nonlinear models. J Rheumatol 1992;19:1655–8. 9 Weyand CM, McCarthy TG, Goronzy JJ. Correlation 2 Nepom GT, Nepom BS. Prediction of susceptibility to between disease phenotype and genetic heterogeneity in rheumatoid arthritis by human leukocyte antigen typing. rheumatoid arthritis. J Clin Invest 1995;95:2120–6. Rheum Dis Clin North Am 1992;18:785–92. 10 Weyand CM, Hicok KC, Conn DL, Goronzy JJ. The influ- 3 Ollier W, Thomson W. Population genetics of rheumatoid ence of HLA-DRB1 genes on disease severity in rheuma- arthritis. Rheum Dis Clin North Am 1992;18:741–58. toid arthritis. Ann Internal Med 1992;117:801–6. 4 MacGregor A, Ollier W, Thomson W, Jawaheer D, Silman 11 Wordsworth P, Pile KD, Buckley JD, Lanchbury JSS, Ollier A. HLA-DRB1*0401/0404 genotype and rheumatoid B, Lanthrop M, et al. HLA heterozygosity contributes to arthritis: increased association in men, young age at onset susceptibility to rheumatoid arthritis. Am J Hum Genet and disease severity. J Rheumatol 1995;22:1032–6. 1992;51:585–91. 5 Gough A, Faint J, Salmon M, Hassell A, Wordsworth P, 12 Balas A, Garcia-Sanchez F, Gomez-Reino F, Vicario JL. Pilling D, et al. Genetic typing of patients with inflamma- HLA class 1 allele (HLA-A2) expression defect associated tory arthritis at presentation can be used to predict with a mutation in its enhancer B inverted cat box in two outcome. Arthritis Rheum 1994;37:1166–70. families. Hum Immunol 1994;41:69–73. 6 Drover S, Marshall WH, Kwok WW, Nepom GT, Karr RW. 13 Beaty JS, West KA, Nepom GT. Functional eVects of a Amino acids in the peptide-binding groove influence an natural polymorphism in the transcriptional regulatory antibody-defined, disease associated HLA-DR epitope. sequence of HLA-DQB1. Mol Cell Biol 1995;15:4771– Scand J Immunol 1994;39:539–50. 82. http://ard.bmj.com/ on September 29, 2021 by guest. Protected copyright.